Background-Suppressed High-Throughput Mid-Infrared Photothermal Microscopy via Pupil Engineering

Area of Science:

• Optics and Photonics
• Chemical Imaging
• Microscopy

Background:

• Mid-infrared photothermal (MIP) microscopy offers label-free chemical imaging with high specificity and resolution.
• Wide-field MIP modalities enhance imaging speed but struggle with sensitivity due to background light overwhelming weak signals from subwavelength particles.
• Limited signal-to-noise ratio hinders the detection of small particles and detailed chemical analysis.

Purpose of the Study:

• To develop a background-suppressed chemical fingerprinting technique for single nanoparticle detection using MIP microscopy.
• To improve the sensitivity and signal-to-background noise ratio (SBNR) in MIP imaging.
• To demonstrate the capability for high-throughput, label-free chemical imaging of various biological samples.

Main Methods:

• Implemented pupil engineering in the collection path to selectively attenuate reflected light, creating quasi-darkfield illumination in an epi-configuration.
• Developed a theoretical framework for photothermal image formation.
• Experimentally validated the technique using polymer beads and applied it to bacteria and cell imaging.

Main Results:

• Achieved over 3 orders of magnitude background suppression without compromising lateral resolution.
• Demonstrated a 6-fold improvement in signal-to-background noise ratio (SBNR).
• Enabled simultaneous detection and discrimination of hundreds of nanoparticles across a 70 μm × 70 μm field of view.

Conclusions:

• The developed background-suppressed MIP technique significantly enhances sensitivity for label-free chemical imaging at the single nanoparticle level.
• The method allows for high-throughput, high-SBNR imaging of diverse biological specimens, including bacteria and subcellular structures.
• This advancement broadens the applicability of MIP microscopy for detailed chemical characterization in various scientific fields.